Complement mediated hepatocytes injury in a model of autoantibody induced hepatitis

Abstract

Despite multiple reports on autoantibody-initiated complement activation in autoimmune hepatitis (AIH), how does the humoral immunity contribute to the pathogenesis of AIH remained unclear. In this report, by adoptively transferring a polyclonal rabbit anti-OVA antibody into Hep-OVA Tg mice in which OVA is selectively expressed on the surface of hepatocytes, we found that excessive complement activation initiated by the autoantibody overwhelmed the protection of intrinsic cell surface complement regulators, and induced hepatocytes injury both in vitro and in vivo. The anti-OVA antibody induced hepatic injury in Hep-OVA Tg but not WT C57BL/6 mice as assessed by serum ALT levels and liver histopathology. Immunohistochemical analyses showed that after the antibody administration, there was massive complement activation on anti-OVA IgG coated hepatocytes in Hep-OVA Tg mice, but not in WT mice. Consistent with these results, depleting complement by cobra venom factor (CVF) prior to antibody injections protected Hep-OVA Tg mice from anti-OVA IgG induced hepatic injury. In addition, treating Hep-OVA Tg mice with recombinant mouse decay accelerating factor, a native complement inhibitor, protected them from autoantibody induced hepatitis. These results suggest that complement could play a pivotal role in liver specific autoantibody mediated hepatocyte injury in AIH, and that complement inhibitors could be, in principle, developed as novel therapeutics against AIH.

Figure 1

Mouse primary hepatocytes express DAF, Crry and CD59. 2 ×10⁵ freshly isolated B6 primary hepatocytes were stained with mAbs against DAF (A), Crry (B) or CD59 (C), followed by flow cytometry analysis. Representative results of three separate experiments.

Figure 2

The hepatocyte-specific antibody activates complement and induces complement mediated hepatocyte injury in vitro. A: 1 ×10⁵ hepatocytes freshly isolated from Hep-OVA mice were incubated with 50 μg/ml anti OVA IgG in MEM media for 20 min. After washing, cells were resuspended in 50 μl GVB²⁺ buffer with 20% normal mouse serum with (dotted line) or without 1 mM EDTA (solid line), and incubated at 37°C for another 30 min. Deposition of complement activation product C3b was detected by staining with 5 μg/ml FITC-anti C3 mAb followed by flow cytometric analysis. Representative results of two separate experiments. B: 1×10⁵ freshly isolated Hep-OVA hepatocytes were labeled with 1 μM BCECF-AM, then incubated with 50 μg/ml anti-OVA IgG and 20% normal mouse serum in GVB²⁺ buffer with (control) or without 1 mM EDTA (experimental) for 30 min. Cell injury was assessed by measuring released BCECF in the superannuates then calculated as described in Methods. The experimental results were normalized against the controls (with EDTA). Combined results from 3 experiments were shown. (* p<0.05)

Figure 3

Administration of the anti-OVA IgG selectively induces complement mediated liver injury in Hep-OVA Tg mice. WT or Hep-OVA Tg mice were i.v. injected with 0.5 mg the anti-OVA IgG and serum ALT levels were measured 4 hrs later. To determine the role of complement in the hepatocyte specific antibody-induced liver injury, complement was depleted in Hep-OVA mice by i.p. injection of 20 μg/mouse CVF, followed by the anti-OVA IgG administration. Each dot represents one mouse and the bars represent the averaged serum ALT values. * p<0.05 between the (Hep-OVA Tg + anti-OVA IgG) group and other groups

Figure 4

Panel A, Representative results of liver histopathological and immunohistochemical analyses of WT (A), Hep-OVA Tg (B) and Hep-OVA Tg mice with CVF complement depletion (C) followed by rabbit anti-OVA IgG administration. Liver samples were collected 4 hr after anti-OVA IgG injections. For H&E stainings, livers were fixed in 4% formaldehyde and 7 μM paraffin-embedded sections were cut and stained (A1, B1 and C1). Arrows indicate infiltrated inflammatory cells. For bound rabbit IgG (A2, B2 and C2), deposited C3b (A3, B3 and C3) and C5b-9 (A4, B4 and C4) staining, livers were snap-frozen in liquid nitrogen and 7 μM cryosections were made. After this, sections were treated with 4% H₂O₂, followed by the anti-rabbit IgG (A2, B2 and C2), anti-C3 mAbs (A3, B3 and C3) and anti C5b-9 Ab (A4, B4 and C4) with an ABC kit. The staining with non-relevant IgGs were negative (not shown). Panel B. Staining of CD11b⁺ infiltrated cells, showing neutrophil-like CD11b⁺ cells in the liver after anti-OVA IgG administration.

Figure 4

Panel A, Representative results of liver histopathological and immunohistochemical analyses of WT (A), Hep-OVA Tg (B) and Hep-OVA Tg mice with CVF complement depletion (C) followed by rabbit anti-OVA IgG administration. Liver samples were collected 4 hr after anti-OVA IgG injections. For H&E stainings, livers were fixed in 4% formaldehyde and 7 μM paraffin-embedded sections were cut and stained (A1, B1 and C1). Arrows indicate infiltrated inflammatory cells. For bound rabbit IgG (A2, B2 and C2), deposited C3b (A3, B3 and C3) and C5b-9 (A4, B4 and C4) staining, livers were snap-frozen in liquid nitrogen and 7 μM cryosections were made. After this, sections were treated with 4% H₂O₂, followed by the anti-rabbit IgG (A2, B2 and C2), anti-C3 mAbs (A3, B3 and C3) and anti C5b-9 Ab (A4, B4 and C4) with an ABC kit. The staining with non-relevant IgGs were negative (not shown). Panel B. Staining of CD11b⁺ infiltrated cells, showing neutrophil-like CD11b⁺ cells in the liver after anti-OVA IgG administration.

Figure 5

Administration of recombinant DAF protein protects anti-OVA-mediated liver injury in Hep-OVA mice. A. E^shA - based C3b uptake assays showing that recombinant DAF protein administration inhibited > 80% of the serum complement activity (mean fluorescence intensity 5 vs 25). B. Administration of recombinant DAF protein significantly reduced serum ALT levels. Each dot represents one mouse and the bars represent the averaged serum ALT values. * p<0.05

Figure 6

Representative H&E and immunohistochemical staining pictures of livers from Hep-OVA Tg mice without (A) and with (B) recombinant DAF protein treatment. A1, H&E stainings of Hep-OVA mouse liver sections 4 hr after anti-OVA IgG administration, showing infiltrated inflammatory cells (arrows); B1, H&E staining of Hep-OVA mouse liver sections with recombinant DAF treatment 4 hr after anti-OVA IgG administration, showing little, if any infiltrated cells; A2, B2, Rabbit IgG staining, showing that rabbit anti-OVA IgG binds to Hep-OVA mouse hepatocytes without (A2) and with (B2) DAF treatments; A3, B3, and A4, B4, complement activation products C3b and C5b-9 staining, showing that C3b accumulation and C5b-9 deposition on hepatocytes are markedly reduced in Hep-OVA Tg mice with DAF treatment (B3, B4) compared to that without DAF treatment (A3, B4). Staining with non-relevant IgGs was negative (not shown).